The beta-2-adrenoreceptor agonists, formoterol and indacaterol, but not salbutamol, effectively suppress the reactivity of human neutrophils in vitro

Abstract

The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories of β 2-agonists, namely, salbutamol (short-acting), formoterol (long-acting) and indacaterol (ultra-long-acting), at concentrations of 1-1000 nM, with human blood neutrophils in vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 µM) or platelet-activating factor (PAF, 200 nM) in the absence and presence of the β 2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of the β 2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca(2+). At the concentrations tested, formoterol and indacaterol caused equivalent, significant (P < 0.05 at 1-10 nM) dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective (P < 0.05 at 100 nM and higher). Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca(2+) from the cytosol of activated neutrophils. These findings demonstrate that β 2-agonists vary with respect to their suppressive effects on activated neutrophils.

Figure 1

Effects of formoterol, indacaterol, and salbutamol (1–1000 nM) on the luminol-enhanced chemiluminescence responses of neutrophils activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 μM). The results are expressed as the mean percentage of control ± SEM (n = 5) with duplicate values for each drug concentration and control system in each experiment. The absolute values for unstimulated neutrophils and for cells activated with fMLP in the absence of the drugs were 2136 ± 219 and 20394 ± 1447 relative light units respectively; *P < 0.04–0.004 for comparison with the fMLP-activated, drug-free control system.

Figure 2

Typical traces from 3 separate experiments (n = 3) showing the effects of formoterol, indacaterol, and salbutamol at 100 nM on the magnitude of oxygen consumption by neutrophils activated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 μM)/cytochalasin B (1 μM) (fMLP/CB).

Figure 3

Effects of formoterol, indacaterol, and salbutamol (1–1000 nM) on the release of elastase from neutrophils activated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 μM)/cytochalasin B (1 μM) (fMLP/CB). The results of 3 separate experiments (n = 3) with 5 replicates for each drug concentration and control system in each experiment are expressed as the mean percentage of control ± SEM. The absolute values for the unstimulated control system and for cells activated with fMLP/CB were 47 ± 6 and 1551 ± 78 milliunits elastase per 10⁷ cells. *P < 0.0102–0.0001 for comparison with the drug-free control systems.

Figure 4

Effects of formoterol, indacaterol, and salbutamol (10 and 100 nM) on the production of LTB₄ by fMLP- (a) and PAF- (b) activated neutrophils. The results of 5 separate experiments (n = 5) with 2 replicates for each drug concentration and control system in each experiment are expressed as the mean percentage of control ± SEM. The absolute values for the unstimulated control system and for cells activated with either fMLP or PAF were 33 ± 7, 1095.4 ± 362.2, and 1546 ± 1108.5 pg mL⁻¹. *P < 0.001 for comparison with the drug-free control systems.

Figure 5

Effects of formoterol, indacaterol, and salbutamol (10 and 100 nM) on cAMP levels in neutrophils. The results of 5 separate experiments (n = 5) with 2 replicates for each drug concentration and control system in each experiment are expressed as the mean ± SEM. The absolute value for the control system was 10.4 ± 0.9 pg mL⁻¹. *P < 0.001 for comparison with the drug-free control systems.

Figure 6

Fura-2 fluorescence traces from 2 representative experiments (n = 4–7 in the series) showing the effects of formoterol, indacaterol, and salbutamol (10 and 100 nM) on the alterations in cytosolic Ca²⁺ concentrations following activation of the cells with either 1 μM fMLP (a) or 200 nM PAF (b). The 3 lines in each trace correspond to the control system (—) and systems treated with either 10 nM (_ _) or 100 nM (●●●●●) of each drug; ↓ denotes the addition of either fMLP or PAF.

Figure 7

Mn²⁺-quenching of fura-2 fluorescence traces from 2 representative experiments (n = 3 in the series) showing the effects of formoterol, indacaterol (100 nM), and salbutamol (100 and 1000 nM) on the alterations in cytosolic Ca²⁺ concentrations following activation of the cells with either 1 μM fMLP (a) or 200 nM PAF (b). The 3 lines in each trace correspond to the control system (—) and systems treated with 100 nM of each drug (●●●●●) or 1000 nM salbutamol (xxxx); ↓ denotes the addition of either fMLP or PAF.